Abstract

Decreased collateral vessel formation in diabetic peripheral limbs is characterized by abnormalities of the angiogenic response
to ischemia. Hyperglycemia is known to activate protein kinase C (PKC), affecting the expression and activity of growth factors
such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF). The current study investigates
the role of PKCδ in diabetes-induced poor collateral vessel formation and inhibition of angiogenic factors expression and
actions. Ischemic adductor muscles of diabetic Prkcd+/+ mice exhibited reduced blood reperfusion, vascular density, and number of small vessels compared with nondiabetic Prkcd+/+ mice. By contrast, diabetic Prkcd−/− mice showed significant increased blood flow, capillary density, and number of capillaries. Although expression of various
PKC isoforms was unchanged, activation of PKCδ was increased in diabetic Prkcd+/+ mice. VEGF and PDGF mRNA and protein expression were decreased in the muscles of diabetic Prkcd+/+ mice and were normalized in diabetic Prkcd−/− mice. Furthermore, phosphorylation of VEGF receptor 2 (VEGFR2) and PDGF receptor-β (PDGFR-β) were blunted in diabetic Prkcd+/+ mice but elevated in diabetic Prkcd−/− mice. The inhibition of VEGFR2 and PDGFR-β activity was associated with increased SHP-1 expression. In conclusion, our data
have uncovered the mechanisms by which PKCδ activation induced poor collateral vessel formation, offering potential novel
targets to regulate angiogenesis therapeutically in diabetic patients.

The main long-term complications from diabetes are vascular diseases, which are in turn the main causes of morbidity and mortality
in diabetic patients (1). Diabetic vascular complications affect several important organs, including the retina, kidney, and arteries (2,3). Peripheral vascular diseases are the major risk factor for nontraumatic lower limb amputation in patients with diabetes
(4), characterized by collateral vessel development insufficient to support the loss of blood flow through occluded arteries
in the ischemic limbs (5). Multiple abnormalities in the angiogenic response to ischemia have been documented in the diabetic state and depend on
complex interactions of multiple growth factors and vascular cells.

Experiments to improve angiogenesis and vascular cell survival by local infusion of vascular endothelial growth factor (VEGF)
or angiopoietin by increasing its expression have also been reported in nondiabetic animal models (6,7). Moreover, animal studies have used platelet-derived growth factor (PDGF) to improve collateral vessel formation and vascular
healing in the diabetic state (8). Clinical trials using recombinant growth factors have noted transient improvement of myocardial and distal leg circulation
(9–11). However, these favorable vascular effects appeared to produce limited clinical benefits (12). Local administration of growth factors, such as VEGF by gene therapy in the setting of diabetes, does not appear to have
the beneficial long-term effects seen in the absence of diabetes or to improve quality of life (13,14). One potential problem with normalizing VEGF or PDGF action alone is that a variety of growth factors may be needed to establish
and maintain the capillary bed.

Various studies have clearly identified that the expression of growth factors, such as VEGF, PDGF, and stromal-derived factor-1
(SDF-1), are critically important in the formation of collateral vessels in response to ischemia (15–17). Previous studies suggested that hyperglycemia attenuates VEGF production and levels in myocardial tissue and in animal
models of wound repair (5,18). Furthermore, decreased VEGF and PDGF expression in the peripheral limbs and nerves of diabetic animals and rodents has
been reported (19–21). Although the underlying mechanism of reduction of VEGF and PDGF expression in diabetes is not clear, it is well-known that
the major inducers of VEGF and PDGF (i.e., hypoxia and oxidants) can both play a role in diabetes. We and other researchers
have reported that variation in PDGF signaling, rather than expression, is linked to morphological abnormalities in the retina
and in collateral capillary formation in an ischemic limb model of diabetic animals (22,23). Clearly, poor collateral vessel formation during diabetes-induced ischemia is attributable to the lack of production and/or
action of critical growth factors such as VEGF and PDGF. Therefore, further studies of the basic mechanisms of hyperglycemia-induced
activation of toxic metabolites, such as activation of protein kinase C (PKC), are needed to identify how these proteins contribute
to growth factor deregulation.

PKC, a member of a large family of serine/threonine kinases, is involved in the pathophysiology of vascular complications.
When activated, PKC phosphorylates specific serine or threonine residues on target proteins that vary, depending on cell type.
PKC has multiple isoforms that function in a wide variety of biological systems (24). PKC activation increases endothelial permeability and decreases blood flow and the production and response of angiogenic
growth factors that contribute to the loss of capillary pericytes, retinal permeability, ischemia, and neovascularization
(25–29).

Previous data have demonstrated that high glucose levels in smooth muscle cells activate PKCα, -β, -δ, and -ε but not the
atypical PKCζ (30,31). In general, high levels of glucose-induced PKC activation cause vascular dysfunction by altering the expressions of growth
factors such as VEGF, PDGF, transforming growth factor-β, and others (32–34). PKCδ has been proposed to participate in smooth muscle cell apoptosis, and deletion of this PKC isoform led to increased
arteriosclerosis (35). Moreover, we previously demonstrated that diabetes-induced PKCδ activation generates PDGF unresponsiveness, causing pericyte
apoptosis, acellular capillaries, and diabetic retinopathy (23). We therefore hypothesized that PKCδ activation could be involved in proangiogenic factor inhibition that triggers poor
collateral vessel formation in diabetes.

Animal and experimental design.

C57BL/6J mice (6 weeks old) were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred in our animal facility. Prkcd−/− mice, described previously and provided by Dr. Michael Leitges (35), were generated by the insertion of a LacZ/neo cassette into the first transcribed exon of the PKCδ gene. This insertion
abolished the transcription of PKCδ, leading to a null allele. Prkcd−/− mice with mixed background of 129SV and C57BL/6J strains were crossbred for 10 generations (F12) with wild-type C57BL/6J
background from The Jackson Laboratory. Animals were rendered diabetic for a 2-month period by intraperitoneal streptozotocin
injection (50 mg/kg in 0.05 mol/L citrate buffer, pH 4.5; Sigma) on 5 consecutive days after an overnight fast; control mice
were injected with citrate buffer. Blood glucose was measured by Glucometer (Contour, Bayer Inc.). Throughout the study period,
animals were provided with free access to water and standard rodent chow (Harlan Teklad, Madison, WI). All experiments were
conducted in accordance with the Canadian Council of Animal Care and University of Sherbrooke guidelines.

Hind limb ischemia model.

We assessed blood flow in nondiabetic and Prkcd+/+ and Prkcd−/− mice diabetic for 2 months. Animals were anesthetized, and the entire lower extremity of each mouse was shaved. A small incision
was made along the thigh all the way to inguinal ligament and extending superiorly toward the mouse abdomen. The femoral artery
was isolated from the femoral nerve and vein and ligated distally to the origin of the arteria profunda femoris. The incision
was closed by interrupted 5-0 sutures (Syneture).

Laser Doppler perfusion imaging and physical examination.

Hind limb blood flow was measured using PIM3 laser Doppler perfusion imaging (Perimed Inc.). Consecutive perfusion measurements
were obtained by scanning the region of interest (hind limb and foot) of anesthetized animals. Measurements were performed
before and after artery ligation and on postoperative days 7, 14, 21, and 28. To account for variables that affect blood flow
temporally, the results at any given time were expressed as a ratio against simultaneously obtained perfusion measurements
of the right (ligated) and left (nonligated) limb. Tissue necrosis was scored to assess mice that had to be killed during
the course of the experiment due to necrosis/loss of toes.

Histopathology and TUNEL assay.

Right and left abductor muscles from Prkcd+/+ and Prkcd−/− mice were harvested for pathological examination. Sections were fixed in 4% paraformaldehyde (Sigma-Aldrich) for 18 h and
then transferred to 90% ethanol for light microscopy and immunohistochemistry. Paraformaldehyde-fixed tissue was embedded
in paraffin, and 6-µm sections were stained with hematoxylin and eosin (Sigma). Apoptotic cells were detected using the TACS
2 Tdt-Fluor in situ apoptosis detection kit (Trevigen, Gaithersburg, MD) according to the manufacturer’s instructions.

Immunofluorescence.

Adductor muscles were blocked with 10% goat serum for 1 h and exposed in sequence to primary antibodies (CD31 and α-smooth
muscle actin, 1:100) overnight, followed by incubation with secondary antibodies Alexa-647 conjugated anti-rabbit IgG and
Alexa-594 conjugated anti-mouse (1:500; Jackson ImmunoResearch Laboratories). Confocal images were captured on a Zeiss LSM
410 microscope; images of one experiment were taken at the same time under identical settings and handled in Adobe Photoshop
similarly across all images.

Real-time PCR analysis.

Real-time PCR was performed to evaluate mRNA expressions of PKCα, PKCβ, PKCδ, PKCε, VEGF, PDGF, KDR/Flk-1, PDGFR-β, endothelial
NOS (eNOS), SDF-1, FGF2, SHP-1, SHP-2, and PTP1B of nonischemic and ischemic limbs. Total RNA was extracted from adductor
muscles with TRI-REAGENT, as described by the manufacturer and RNeasy mini kit (Qiagen, Valencia, CA). The RNA was treated
with DNase I (Invitrogen) to remove any genomic DNA contamination. Approximately 1 μg RNA was used to generate cDNA using
SuperScript III reverse transcriptase and random hexamers (Invitrogen) at 50°C for 60 min. PCR primers and probes are listed
in Supplementary Table 1. Glyceraldehyde-3-phosphate dehydrogenase and 18S ribosomal RNA expression were used for normalization. PCR products were
gel purified, subcloned using a QIA quick PCR Purification kit (Qiagen), and sequenced in both directions to confirm identity.

Nuclear extract and nonradioactive transcription factor assay.

Adductor muscles were lysed and nuclear-specific proteins isolated using the NucBuster Protein Extraction Kit (Novagen, Madison,
WI) according to the manufacturer’s instructions. Detection of hypoxia-inducible factor-1α (HIF-1α) in the nucleus was quantified
using the nonradioactive transcription factor assay kit (Cayman, Ann Arbor, MI). Briefly, nuclear protein (20 µg) was incubated
for 24 h in a 96-well plate containing immobilized specific double-stranded DNA consensus sequence of the HIF-1α response
element. HIF-1α contained in the nuclear extract was linked specifically to the HIF-1α response element. Wells were washed
five times, and the HIF transcription factor complex was detected by addition of a specific primary antibody directed against
HIF-1α and incubated for 1 h. Wells were washed five times and exposed with secondary antibody conjugated to HRP for 1 h.
Wells were then washed five times, and developing agent was added to provide a sensitive colorimetric readout at 450 nm (Infinite
M200; Tecan Group Ltd., Männedorf, Switzerland) to quantify nuclear HIF-1α levels.

Statistical analyses.

The data are shown as mean ± SD for each group. Statistical analysis was performed by unpaired t test or by one-way ANOVA, followed by the Tukey test correction for multiple comparisons. All results were considered statistically
significant at P < 0.05.

One main effect of hypoxia is to induce angiogenesis and to promote new capillary formation. To test whether activation of
PKCδ is responsible for poor collateral vessel formation in diabetes, we measured capillary density and capillary diameter
in the ischemic adductor muscles. Figure 3 demonstrated that the adductor muscles of diabetic Prkcd+/+ mice displayed a 31% vascular density reduction compared with nondiabetic Prkcd+/+ mice. The decline of capillary density was accompanied with a 50% reduction in number of vessels with a diameter of 50 µm
or less. Interestingly, diabetic Prkcd−/− mice showed a significant increase in capillary density and number of vessels with a diameter of less than 50 µm compared
with diabetic Prkcd+/+ mice (Fig. 3D).

Histological and vascular density analysis. Structural analysis of the ischemic muscles stained with hematoxylin and eosin
(A) and immunofluorescence of endothelial cells (red) and α-smooth muscle actin (green) labeling (B) in the ischemic adductor muscles of nondiabetic (NDM) and diabetic (DM) Prkcd+/+ and Prkcd−/− mice. Quantification of the vascular density (C) and the number of vessels smaller than 50 µm (D). Results are shown as mean ± SD of three sections of six mice per group.

PKCδ is activated in diabetic ischemic limb.

Hyperglycemia is known to activate multiple PKC isoforms, preferably the β and δ isoforms, in vascular cells. Expression of
various isoforms of PKC was assessed by quantitative PCR in muscle tissues (Fig. 4). Compared with nondiabetic Prkcd+/+ mice, mRNA expression of PKCβ and δ was modestly increased in adductor muscles of diabetic Prkcd+/+ mice and unchanged in Prkcd−/− mice (Fig. 4B and D). There was no significant difference in the mRNA expression of PKCα and -ε (Fig. 4A and C). Although diabetic Prkcd+/+ mice did not exhibit higher levels of protein expression of PKCα, -β2, -ε, or -δ isoforms, adductor muscles of Prkcd+/+ mice showed a significant and impressive increase of PKCδ phosphorylation (Thr 505; P = 0.0040), as a marker of PKCδ activation, 28 days after unilateral femoral artery ligation compared with nondiabetic Prkcd+/+ mice (Fig. 5).

To explain how the absence of PKCδ improved reperfusion in diabetic ischemic limbs, we performed a wide analysis of the gene
and protein expression of angiogenic-related factors and their receptors. Quantitative gene expression analyses by real-time
PCR indicated that VEGF-A, PDGF-B, and PDGFR-β mRNA expression was significantly decreased in the adductor muscles of diabetic
mice by 46, 30, and 63%, respectively, compared with nondiabetic Prkcd+/+ mice (Fig. 6A, C, and D). The reduction of these genes in diabetic Prkcd+/+ mice was not observed in diabetic Prkcd−/− mice. Moreover, mRNA expression of VEGFR2 (KDR/Flk-1), PDGF-B, and PDGFR-β was significantly upregulated in diabetic Prkcd−/− compared with diabetic Prkcd+/+ mice (Fig. 6B–D). These results suggest that impaired PDGF and VEGF expression by PKCδ activation might be the contributing factor for poor
collateral vessel formation in diabetes. Expression of other angiogenic factors, such as SDF-1, FGF-2, and eNOS, as well as
transcriptional factor activity of HIF-1α, was unchanged within all groups of mice (Fig. 6E–H and Supplementary Fig. 1). In contrast to 4 weeks after femoral artery ligation, transcriptional factor activity and mRNA levels of HIF-1α were significantly
decreased in diabetic Prkcd+/+ mice compared with nondiabetic Prkcd+/+ and diabetic Prkcd−/− mice (Supplementary Figs. 2 and 3).

VEGFR2 and PDGFR-β activation is decreased in diabetic ischemic muscles.

To further investigate the mechanisms of impaired angiogenic response to restore blood flow in diabetes, the expression, activation,
and signaling pathway of VEGF-A and PDGF-B and their respective receptors (VEGFR2 and PDGFR-β) were examined. Protein expression
of PDGF-B was significantly decreased in diabetic versus nondiabetic adductor muscles of wild-type animals. In contrast, VEGF-A
and PDGF-B protein expression was elevated in the ischemic limb of the diabetic PKCδ null mice (Fig. 7A and B). Phosphorylation of VEGFR2 and PDGFR-β was inhibited in ischemic adductor muscles of diabetic mice compared with nondiabetic
Prkcd+/+ mice. However, activation of Src was elevated in adductor muscles of diabetic Prkcd+/+ mice compared with nondiabetc Prkcd+/+ and Prkcd−/− mice (Fig. 7B). Interestingly, tyrosine phosphorylation of VEGFR2 and PDGFRβ, as well as PLCγ1, Akt, and ERK phosphorylation, was greatly
enhanced in Prkcd−/− mice compared with diabetic Prkcd+/+ mice (Fig. 7A and B). We did not observe any changes in the eNOS protein expression among experimental groups (Fig. 7A).

Expression of SHP-1 caused VEGFR2 and PDGFR-β inactivation.

We have previously shown that activation of PKCδ leads to increased expression of SHP-1, which inhibits the PDGF-signaling
pathway and promotes retinal pericyte apoptosis in diabetic animals. To determine whether SHP-1 is implicated in PKCδ-induced
VEGFR2 and PDGFR-β dephosphorylation in diabetic ischemic adductor muscles, we measured SHP-1 expression by quantitative PCR
and immunoblot analysis. Figure 8A and B indicates that mRNA expression of SHP-1, but not SHP-2 or PTP1B, is elevated in diabetic Prkcd+/+ mice, whereas SHP-1 is clearly downregulated in Prkcd−/− mice. We confirmed through immunoblot analysis that SHP-1 protein expression was elevated by 2.3-fold in ischemic adductor
muscles of diabetic Prkcd+/+ mice compared with nondiabetic Prkcd+/+ mice. The increase expression of SHP-1 was not observed in diabetic Prkcd−/− mice (Fig. 8C). No change was detected in the protein expression of SHP-2 and PTP1B within all groups of mice (Fig. 8D).

DISCUSSION

Diabetes is associated with the progression of vascular complications, such as peripheral arterial disease, and is a major
risk factor for lower limb amputations (4). In the current study, we have demonstrated that activation of PKCδ diminishes the expression of VEGF and PDGF, two critical
proangiogenic factors contributing to poor capillary formation and blood flow reperfusion of the ischemic limbs. In addition
to reducing expression of VEGF and PDGF, phosphorylation of VEGF and PDGF receptors was abrogated in diabetic ischemic muscles
compared with nondiabetic ischemic muscles. The inhibition of growth factor receptor phosphorylation was associated with the
upregulation of SHP-1 expression, which has been reported to deactivate tyrosine kinase receptors such as VEGF and PDGF receptors.
Overall, deletion of PKCδ prevents the reduction of VEGF and PDGF expression and re-establishes KDR/Flk-1 and PDGFR-β phosphorylation,
favoring new capillary formation and blood flow reperfusion.

Wound healing is a complex, well-orchestrated, and dynamic process that involves a coordinated and precise interaction of
various cell types and mediators. Given the fundamental contribution of VEGF and PDGF to the angiogenic process, the mechanism
by which activation of PKCδ isoform prevents growth factors expression and signaling actions may provide a better understanding
of how diabetes reduces collateral vessel formation in the ischemic limb. In this study, we demonstrated that PKCδ is activated
in diabetic ischemic muscles and reduced blood flow reperfusion, contributing to tissue necrosis, amputation, and apoptosis.
Previous studies have reported that PKCδ is involved in vascular cell apoptosis. PKCδ activates p-38, mitogen-activated protein
kinase, p53, and caspase-3 cleavage to favor endothelial (36) and smooth muscle cell apoptosis (37,38). Therefore, deletion of PKCδ may enhance vascular cell migration and proliferation, two significant steps in the formation
of new blood vessels.

Total expression of PKC isoform in ischemic muscles was slightly affected by diabetes, probably because mRNA and protein analyses
were performed 28 days after femoral artery ligation. However, phosphorylation of PKCδ on threonine 505, a phosphorylation
site within the activation loop, clearly suggests that PKCδ is activated in the muscles of diabetic ischemic limbs compared
with nondiabetic muscles. Previous data showed that the inhibition of PKCδ, using an isozyme-specific peptide, improved the
number of microvessels and cerebral blood flow after acute focal ischemia in normotensive rats (39). Our data demonstrate that deletion of PKCδ restores blood flow perfusion in diabetic ischemic muscles by promoting the
number of capillaries and reducing tissue apoptosis.

The reduction of VEGF and PDGF receptor expression and the downstream signaling pathway is associated with impaired angiogenesis
process in diabetic foot ulcer and ischemic diseases. Our results indicate that diabetes-induced PKCδ activation decreases
VEGF, PDGF, KDR/Flk-1, and PDGFR-β mRNA expression in the ischemic limb, which is completely restored in PKCδ-null mice. Interestingly,
impaired angiogenic response in ischemic arterial diseases of type 1 and type 2 diabetes is associated with VEGF inhibition
in endothelial cells and monocytes (13,40). It is possible that the ablation of PKCδ may also affect VEGF signaling in monocytes, which may contribute to vessel formation
abnormalities. However, this assumption will need further investigation.

HIF-1α is a master regulator of angiogenic factors in response to tissue hypoxia. Previous study showed that HIF-1α gene transfer
increased recovery of limb perfusion, increasing eNOS activation and vessel density (41). In our study, however, the increase in the expression of VEGF in muscles of PKCδ-deficient mice may not have been entirely
due to upregulation of HIF-1α. Because protein extraction was performed 4 weeks after the femoral artery ligation, it is possible
that the expression of HIF-1α could have returned to basal levels. This hypothesis is supported by results obtained 2 weeks
after the surgery. Our data demonstrated that HIF-1α transcriptional factor activity and mRNA expression were increased in
nondiabetic and diabetic PKCδ-null mice 2 weeks only after surgery (Supplementary Figs. 2 and 3). Besides VEGF and PDGF expression, our data suggest that PKCδ activation disrupts VEGF and PDGF signaling, whereas in PKCδ-deficient
mice, the activity of VEGFR2, PDGFR-β, PLCγ1, Akt, and ERK is enhanced. Surprisingly, Src phosphorylation was increased in
the ischemic muscles of diabetic wild-type mice even if PDGFR-β activity was reduced. However, a previous study reported that
reactive oxygen species (ROS) production induced Src phosphorylation (42). Because ROS are massively produced in ischemic and hyperglycemic conditions, it is probable that ROS production is responsible
for the Src phosphorylation seen in diabetic wild-type mice.

There is strong evidence that progenitor cell recruitment and homing participate in angiogenesis and wound repair, which are
guided by SDF-1 (43). Although the number of progenitor cells is reduced in diabetic mice, inadequate progenitor cell mobilization has been proposed
as one potential mechanism of impaired angiogenesis (44). However, our results did not observe any change in SDF-1 expression in PKCδ-null mice, suggesting that mobilization and
local trafficking of progenitor cells to the ischemic site was not affected by the PKCδ isoform.

Despite advances in revascularization techniques, limb salvage and pain relief cannot be achieved in many diabetic patients
with diffuse peripheral vascular disease. VEGF-mediated gene therapy has shown promising results as an innovative method in
the treatment of severe cardiovascular diseases. However, a randomized study of gene therapy failed to meet the primary objective
of significant amputation reduction (45). During the 10-year follow-up period, no significant differences were detected in the number of amputations or causes of
death with the use of transient VEGF-A–mediated gene therapy. One reason for this lack of improvement is perhaps because neovascularization
requires the interaction of multiple growth factors that can promote, in a synergic manner, new and mature blood vessels.
Enhancing the responsiveness of diabetic vascular cells to proangiogenic factors may offer a potential new approach to treat
peripheral arterial diseases. Protein tyrosine phosphatase is a group of proteins that is critical in abating cell response
to growth factors by inhibiting tyrosine kinase phosphorylation. Our results demonstrated that SHP-1 expression was increased
in diabetic ischemic muscles and was responsible for VEGF and PDGR receptor dephosphorylation.

Although not significant, a slight rise in SHP-2 (18%) and PTP1B (37%) expression was observed in diabetic PKCδ-null mice.
Previous studies have shown that PDGF activation enhanced SHP-2 and PTP1B activity (46,47), which may explain our results. We have reported that activation of PKCδ induces the expression of SHP-1 in cultured pericytes
exposed to high glucose concentrations and inhibits the PDGF signaling pathway contributing to pericyte apoptosis (23). Others studies have also shown that SHP-1 is a negative regulator of VEGF signal transduction and inhibits endothelial
cell proliferation (48,49). Interestingly, silencing SHP-1 increased phosphorylation of KDR/Flk-1 and markedly enhanced capillary density in a nondiabetic
hind limb ischemia model (50). However, our current study does not provide a direct link between SHP-1 expression and reduced angiogenesis, which will
require further investigations. Nevertheless, our findings have identified PKCδ, and potentially SHP-1, as potential therapeutic
targets for the treatment of diabetic peripheral arterial diseases and cardiovascular complications.

In summary, we have provided evidence that PKCδ is activated by diabetes in ischemic muscles and induced SHP-1 expression,
contributing to VEGF and PDGF unresponsiveness and poor angiogenesis response. Although various therapies are partly successful
in restoring blood flow to the affected tissues, there is no effective strategy to specifically produce new functional vessels
to dismiss diabetic ischemic stress. Our data enhance our understanding of the mechanisms underlying poor collateral vessel
formation induced by PKC activation and may offer potential novel targets to regulate angiogenesis therapeutically in patients
with diabetes.

ACKNOWLEDGMENTS

This study was supported by grants from the Canadian Diabetes Association, Fonds de Recherche du Québec–Santé, and Diabète
Québec to P.G. and was performed at the Centre de Recherche Clinique Étienne-Le Bel, a research center funded by the Fonds
de Recherche du Québec–Santé. P.G. is currently the recipient of a Scholarship Award from the Canadian Diabetes Association
and the Canadian Research Chair in Vascular Complications of Diabetes.

No potential conflicts of interest relevant to this article were reported.

F.L., M.P., B.D., and P.G. performed experiments and analyzed the data. M.L. provided the Prkcd-deficient mice. A.G. performed animal care and researched data. F.L. and P.G. wrote the manuscript. P.G. is the guarantor
of this work, and, as such, had full access to all the data in the study and takes responsibility for the integrity of the
data and the accuracy of the data analysis.

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